The invention relates to a method for influencing the miR-92 expression in a cell, in particular for promoting the angiogenesis, vascularization or vessel repair, or for inhibiting or blocking tumor angiogenesis, and the use of such a method for the therapy of a disease or a condition. Furthermore, the invention relates to a pharmaceutical composition for promoting angiogenesis, vascularization or vessel repair, or for inhibiting or blocking tumor angiogenesis in a cell.
The endothelium plays an important role in the maintenance of the integrity and functionality of the vessels. In the adult, the growth of new vessels occurs through arteriogenesis, angiogenesis or vasculogenesis. Whilst arteriogenesis is defined as the growth of collateral vessels, angiogenesis is understood as the growth of new blood vessels from pre-existing vessels. During angiogenesis, resting endothelial cells are activated by angiogenic factors and start to migrate, proliferate and organize themselves into tubular structures (2). The term vasculogenesis initially described the de novo blood vessel formation in the embryo from angioblasts, but now also includes the formation of blood vessels from endothelial precursor cells or other adult stem cells (1). Angiogenesis and vasculogenesis represent physiological developmental processes which play an essential role in the reconstitution of the blood flow in ischemic tissues, and are a basic step in the growth of tumors. The promotion of angiogenesis and neovascularization was identified as a possible therapeutic strategy in, for example, patients suffering from ischemia. In tumor angiogenesis, the limitation of these processes leads to a repression of tumor growth.
MicroRNAs (miRNAs) are small non-encoding RNAs that regulate the gene expression on the post-transcriptional level through a degradation of the target mRNA or through translational repression (3). In contrast to small interfering RNAs (siRNA), which bind to complementary mRNA sequences, the binding of the miRNA to a target does not only take place at a complementary RNA, but forms more complex RNA-RNA-structures that are thermodynamically preferred (4). This “incomplete” binding allows the binding of one miRNA molecule to different mRNA molecules. The regulation of a set of genes (in contrast to a monotherapy using a gene or a growth factor) can represent an advantage, if thereby complex regulatory processes can be influenced. So far, more than 400 miRNAs have been identified in the human genome, but the relevance of most of these miRNAs for the cellular function in physiological and pathologic processes is still unclear. Whereas it was described in the state of the art that the down-regulation of the miRNA-processing enzymes Dicer and Drosha impedes angiogenesis (5-7), only few specific miRNAs have been described which influence endothelial cell functions and angiogenesis. MiR-221 and miR-222 block the endothelial cell migration, proliferation and angiogenesis in vitro in an indirect manner through an interaction with the stem cell factor receptor c-kit and regulation of the eNOS expression (6, 8). In contrast to this, the expression of let7-f and miR-27b contributes to an in vitro angiogenesis (7).
The state of the art attributed a strong tumor angiogenesis-promoting activity to the miRNA cluster miR-17-92. The miR-17-92 cluster consists of miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92-1 (9). This miR-17-92 cluster is up-regulated in Myc-induced tumors, and the individual miRNAs miR-18 and miR-19 could be identified as molecules which specifically interact with the expression of anti-angiogenic proteins. A specific evaluation of the targets of these miRNAs showed that miR-18 preferably suppresses the expression of the connective tissue growth factor (CTGF), whereas miR-19 interacts with the strong angiogenesis-inhibitor thrombospondin-1 (TSP-1) (10).
It was surprisingly found in accordance with the invention that miR-92 does not promote the angiogenesis as described in the state of the art, but strongly reduces the migration and tube formation of endothelial cells in vitro and the neovascularization in vivo. The inhibition of miR-92 increases neovascularization. Accordingly, miR-92 has an anti-angiogenic activity and not a pro-angiogenic activity as described so far in the state of the art.
This anti-angiogenic activity is connected to the inhibition of key proteins that control angiogenesis and endothelial activity, including the endothelial nitric oxide synthase (eNOS), and sir-tuin1 (SIRT1), which are both essential for postnatal endothelial cell functions (11 to 13), and integrin a5, which controls the endothelial cell motility and the interaction with the matrix (14). The inhibition of miR-92 by miR-92 inhibitors represents a new therapeutic strategy for improving the endothelial cell function and neovascularization. At the same time, it was found that the over-expression of miR-92 reduces neovascularization.